Modified adenoviral fiber and uses

ABSTRACT

The present invention relates to a modified fiber of an adenovirus, comprising at least one mutation at one or more residues within the region of said fiber stretching from pleated sheet A to pleated sheet B, and including loop AB.

[0001] The present invention relates, in particular, to an adenoviralfiber mutated in the regions involved in recognizing and binding to thenatural cellular receptor for adenoviruses. It also relates to theadenoviral particles bearing, at their surface, such a fiber, optionallycombined with a ligand which confers modified, or even targeted, hostspecificity on said particles. The invention is of most particular valuein the context of the development of vectors which can be used in thecontext of gene therapy.

[0002] Adenoviral vectors are widely used in many gene therapyapplications. They have been demonstrated in many animal species and arerelatively nonpathogenic, and nonintegrating, and replicate both individing and in quiescent cells. In addition, they have a broad hostspectrum and are capable of infecting a very great number of cell types,such as for example epithelial cells, endothelial cells, myocytes,hepatocytes, nerve cells and synoviocytes (Bramson et al., 1995. Curr.Op. Biotech. 6, 590-595).

[0003] The adenoviral genome consists of a double stranded, linear DNAmolecule of approximately 36 kb containing two inverted repeat regions(referred to as ITRs for Inverted Terminal Repeat) framing the genesencoding the viral proteins. The early genes are divided into fourregions dispersed in the adenoviral genome (E1 to E4; E for early),including 6 transcriptional units provided with their own promoters. Thelate genes (L1 to L5; L for late) cover, in part, the earlytranscription units and are, mostly, transcribed from the major latepromoter (MLP).

[0004] Adenoviruses have been the subject of many studies and manyscientific teams have developed adenoviral vectors which arereplication-defective, i.e. in which the genome has been manipulatedsuch that these adenoviral vectors are incapable of dividing or ofproliferating in the cells which they infect. Defective adenoviralvectors are in particular obtained by deleting at least the El region(for examples of defective adenoviral vectors, see, in particular,patent applications WO 94/28152 and WO 94/12649).

[0005] More recently, other uses of adenoviral particles have beendescribed, in particular in the context of implementing gene therapyprotocols.

[0006] Thus, patent application WO 95/21259 describes a method forintroducing a nucleic acid into a cell, which is based on combiningadenoviral particles and nucleic acid, more particularly naked nucleicacid. This method is based mainly on the capacity of the adenoviralparticle to transport molecules to the cell nucleus' after endocytosis.Curiel et al. (1992 Hum. Gene Ther., 3: 147-154) and Wagner et al.(1992, Proc. Natl. Acad. Sci., 89; 6099-6103), have, themselves also,shown that combining plasmid with inactivated adenoviral particlesallows the endosome to be lysed before fusion with the lysosomes and,therefore, allows the plasmid to escape degradation. This ingeniousdevice makes it possible to increase the efficiency of transfection ofthe plasmid 100- to 1000-fold in vitro. Preferably, in order for thecellular transfection to be independent of the adenoviral process and toindeed involve the use of a ligand chosen so as to allow targeting ofthe transfection, an antibody which neutralizes the adenoviral infectioncan be added to the complex (Michael et al.; 1993, J. Biol. Chem., 268:6866-6869). The contents of these publications and patent applicationsare incorporated by reference in their entirety, into the presentapplication.

[0007] The infectious cycle of adenoviruses is based on two essentialsteps. The early phase precedes replication initiation and allows theproduction of the early proteins which regulate replication andtranscription of the viral DNA. Replication of the genome is followed bythe late phase during which the structural proteins which constitute thebasis of the viral particles are synthesized. Assembly of the newvirions takes place in the nucleus. Initially, the viral proteinsassemble so as to form empty capsids of icosahedrai structure, in whichthe newly formed genome is encapsidated. The adenoviruses released arecapable of infecting other permissive cells.

[0008] During infection, the fiber and the penton base of the adenoviralparticle, present at the surface of the capsids, play a critical role inthe cellular attachment of the virions and their internalization(Wickham et al., 1993, Cell, 73, 309-319). Firstly, the adenovirus bindsto a cellular receptor (the CAR) present at the surface of thepermissive cells, via the fiber in its trimeric form (Philipson et al.,1968, J. Virol. 2, 1064-1075; Defer et al., 1990, J. Virol, 64,3661-3673). The viral particle is then internalized by endocytosis, dueto binding of the penton base to the α_(v)β₃ and α_(v)β₅ cellularintegrins (Mathias et al., 1994, J. Virol. 68, 6811-6814).

[0009] The adenoviral fiber is composed of three distinct domains(Chroboczek et al., 1995, Current Top. Microbiol. Immunol. 199,165-200):

[0010] (a) at its N-terminal end, is the tail, the sequence of which isvery conserved from one adenoviral serotype to the other. It interactswith the penton base and ensures the anchoring of the molecule in thecapsid;

[0011] (b) in the center, is the shaft. It is a rod-like structurecomposed of a certain number of pleated-sheet repeats, the number ofwhich varies according to the serotypes under consideration;

[0012] (c) at its C-terminal end is the Knob, which has a sphericalglobular structure containing the trimerization signals (Hong andEngler, 1996, J. Virol. 70, 7071-7078; Novelli and Boulanger, 1991, J.Biol. Chem. 266, 9299-9303; Novelli and Boulanger, 1991, Virology 185,365-376), and is responsible for the binding to permissive cells (Henryet al., 1994, J. Virol 68, 5239-5246; Louis et al., 1994, J. Virol. 68,4104-4106).

[0013] Several teams have already described adenoviral particles forwhich the native fiber has been modified so as to modify their naturaltropism and change the binding specificity of this fiber such that itrecognizes a different cellular receptor.

[0014] WO 94/10323 describes type 5 (Ad5) adenoviral particles in whichthe fiber has been mutated so as to comprise the sequence of a fragmentof antibody specific for a given antigen (of scFv type), inserted at theend of one of the 22 repetitive units of the shaft. These mutants have amodified specificity of infection of the adenoviral particles and arecapable of attaching to cells exhibiting the target antigen.

[0015] U.S. Pat. No. 5,543,328 describes a chimeric adenoviral fiber inwhich the Knob domain is replaced with the tumor necrosis factor (TNF)sequence, or that of the ApoE peptide, so as to redirect the attachmentof the modified adenoviral particles toward cells expressing thecellular receptor for TNF or the LDL (low density lipoprotein) receptor,respectively, present at the surface of hepatic cells.

[0016] WO 95/26412 describes a fiber modified by incorporating a ligandat the C-terminal end.

[0017] WO 96/26281 describes a chimeric fiber obtained by replacing aportion of the native fiber, and in particular of the knob, with theequivalent portion of an adenoviral fiber of another serotype and,optionally, inserting a vitronectin-specific RGD peptide at theC-terminal end.

[0018] In addition, French patent application FR 2758821 (97 01005) hasdemonstrated the role of the class I major histocompatibility complexantigens and of the III modules of fibronectin as a primary receptor anda cofactor, respectively, for adenoviruses. In an identical way, Tomkoet al. (1997, Proc. Natl. Acad. Sci 94, 3352-3356), Bergelson et al.(1997, Science 275, 1320-1323) and Roelvink et al. (1998, J. Virol. 72,7909-7915) have described another receptor for the fiber of variousadenoviral serotypes. It is a 46 kDa surface molecule, CAR (Coxsackieand Adenovirus Receptor).

[0019] Finally, Xia et al. (1994, Structure 2, 1259-1270) havedetermined the crystallographic three-dimensional structure of theadenoviral knob. Each monomer includes 8 antiparallel β-pleated sheets,referred to as A to D and G to J, and 6 major loops of 8 to 55 residues.For example, loop CD connects pleated sheet C to pleated sheet D. It isindicated that minor pleated sheets E and F are considered to form partof loop DG located between pleated sheets D and G. By way of indication,Table 1 gives the location of the structures in the amino acid sequenceof the Ad5 fiber, as shown in sequence identifier No. 0.1 (SEQ ID NO:1), the +1 representing the Met initiating residue. In general, thepleated sheets form an organized and compact structure, whereas theloops are more flexible. These terms are conventional in the field ofprotein biochemistry, and are defined in fundamental works (see, forexample, Stryer, Biochemistry, 2nd Edition, Chap. 2, p. 11 to 39, EdFreeman and Company, San Francisco). TABLE 1 β pleated sheet Loopnomenclature Residues nomenclature Residues A 400 to 403 AB 404 to 418 B419 to 428 — — C 431 to 440 CD 441 to 453 D 454 to 461 DC 462 to 514 G515 to 521 GH 522 to 528 H 529 to 536 HI 537 to 549 I 550 to 557 IJ 558to 572 J 573 to 578

[0020] The four β-pleated sheets A, B, C and J constitute the V pleatedsheets directed toward the viral particle. The other four (D, G, H andI) form the R pleated sheets, which are presumed to face the cellularreceptor. The V pleated sheets seem to play an important role in thetrimerization of the structure, while the R pleated sheets are thoughtto be involved in the interaction with the receptor.

[0021] The present invention provides novel mutants of the adenoviralfiber which allow, in particular, the production of viral particleswhich have the following properties:

[0022] (i) the adenoviral particle comprising said mutated fiber doesnot substantially attach to the natural cellular receptors, i.e. thehost specificity of these adenoviral particles bearing the mutated fiberis decreased, or even inhibited, in comparison to the host specificityof the adenoviral particles carrying the wild-type, i.e. nonmutated,fiber;

[0023] (ii) when the adenoviral particle comprising said mutated fiberalso comprises a ligand specific for an antiligand, it is possible toconfer on said modified particle a novel tropism for one or morespecific cell types bearing, at its (their) surface, said antiligand, incomparison to the nonmutated adenoviral particle.

[0024] The expression “the mutated fiber does not substantially attachto the natural cellular receptors” is intended to indicate that thefiber is modified so as to decrease or abolish its ability to bind tothe natural cellular receptor. Such a property can be verified bystudying the infectivity or the cellular binding of the correspondingadenoviral particles, using the techniques of the art, and, inparticular, with infection competition experiments for the virus bearingthe modified fiber, carried out in the presence of a competitorconsisting of all or part of the wild type adenoviral fiber (for moredetail relating to this measuring technique, see the ExperimentalSection of the present application). The loss of the natural specificitycan also be evaluated with cell attachment studies carried out in thepresence of labeled viruses (for example labeled with ³H thymidine,according to the technique of Roelvink et al., 1996, J. Virol. 70,7614-7621) or with studies of infectivity of permissive cells or ofcells expressing the surface molecule targeted by the ligand (see theexamples which follow). Advantageously, “a mutated fiber does notsubstantially attach to the natural cellular receptors” when thepercentage of residual infection, measured with a competition experimentas disclosed in the examples which follow, is between approximately 0and 60%, preferably between 0 and 40%, and entirely preferably between 0and 20%. In addition, according to an advantageous embodiment, theproperties of trimerization and of binding to the penton-base of themutated adenoviral fiber are not affected. These properties are easilyverified according to the technique used in the examples which follow.

[0025] The present invention has, in particular, the advantage ofproviding novel products, the properties of which make it possible todecrease the therapeutic amounts of adenovirus to be administered and totarget the infection of the vector to the cells to be treated. Thisspecificity is particularly essential when an adenoviral vector is usedwhich is capable of expressing a cytotoxic gene, in order to avoid thepropagation of the cytotoxic effect to the healthy and nontargetedcells. In addition, the teachings of the present invention allow thedevelopment of other targeting systems intended for developing methodsof treatment by administration of recombinant viral or nonviral vectors.

[0026] Firstly, the present invention relates to the modified fiber ofan adenovirus, comprising at least one mutation at one or more residueswithin the region of said fiber stretching from pleated sheet A topleated sheet B, and including loop AB. More particularly, the mutationsare preferably produced at one or more residues within loop AB.

[0027] For the purposes of the invention, the terms “residues” and“amino acids” are synonyms. The terms “pleated sheets” and “loops” aredefined according to Xia et al. 1994, Structure 2, 1259-1270.

[0028] The term “nucleic acid sequence” is intended to refer to asynthetic or isolated natural, linear or circular, double-stranded orsingle-stranded fragment of DNA and/or of RNA and/or PNA which refers toa specific series of nucleotides, which may or may not be modified,making it possible to define a fragment or a region of a nucleic acidwithout size limitation.

[0029] According to a preferred embodiment, it is a nucleic acid chosenfrom the group consisting of a cDNA (complementary DNA); a genomic DNA;a plasmid DNA; an RNA and a viral genome.

[0030] The term “portion” of an amino acid sequence is intended to meanan amino acid sequence comprising a minimum of 6 consecutive aminoacids, preferably 10, more preferably 15, even more preferably 20, andmost preferably 30, and/or having the same biological activity as thesequence from which said portion is derived, in particular the abilityto recognize and to bind to the target cells of the virus.

[0031] The term “portion” of a nucleic acid sequence is intended to meana nucleic acid sequence comprising a minimum of 18 consecutivenucleotides, preferably 30, more preferably 45, even more preferably 60,and most preferably 90, and/or encoding an amino acid sequence havingthe same biological activity as the amino acid sequence encoded by thenucleic acid sequence from which said portion is derived.

[0032] The fiber according to the present invention can derive from anadenovirus of human, canine, avian, bovine, murine, ovine, porcine orsimien origin, or be hybrid and comprise fragments of diverse origins,including fragments of heterologous origin, i.e. not derived from anadenoviral fiber, or derived from nonadenoviral fibers. With regard tohuman adenoviruses, those of serotype C and, in particular, type 2 or 5adenoviruses (Ad2 or Ad5) are preferably used. The fiber of Ad2 includes580 amino acids (aa), the sequence of which is disclosed in Herisse etal. (1981, Nucleic Acid Res. 9, 4023-4042, incorporated into the presentapplication by reference). That of Ad5 has been determined by Chroboczekand Jacrot (1987, Virology, 161, 549-554, incorporated by reference) andhas 582 amino acids (see sequence identifier 1; SEQ ID NO: 1). In orderto simplify the presentation of the present application, only thepositions relating to Ad5 are given. However, it is within the scope ofthose skilled in the art to identify the equivalent positions of thevarious pleated sheets and loops on the basis of the sequences ofadenoviral fibers of other origins. When the fiber of the presentinvention is of animal origin, use is preferably made of bovineadenoviruses and, in particular, those of the BAV-3 strain. The latterhave been the subject of many studies, and the sequence of the fiber isdisclosed in international application WO 95/16048, the content of whichis incorporated by reference. Of course, the fiber of the presentinvention can, besides the modifications described in the presentinvention, have other modifications with respect to the native sequence,as long as they do not affect the characteristics of the fiber proposedin the application. In addition, it is within the scope of those skilledin the art to identify the adenoviral fiber sequences available ondatabases such as, for example, GenBank, and to identify the equivalentpositions of the various pleated sheets and loops as described later. Byway of information, mention is made, for example, of the GenBankreferences for the adenoviral fiber sequences of human serotype 2 (#AAA92223), 3 (# CAA26029), 5 (#M18369), 31 (#CAA54050 or 41 (#X17016).The contents of the publications or of the GenBank references mentionedabove are incorporated, in their entirety, into the present applicationby reference. The invention also relates to a modified fiber accordingto the present invention, which also contains other mutations, such asfor example those described in patent application WO 98/44121. Moreparticularly, such a fiber according to the invention is characterizedin that it also comprises one or more mutations in:

[0033] a) loops CD, DG, GH, HI and/or IJ and/or

[0034] b) pleated sheets C, D, G, H, I and/or J.

[0035] For the purpose of the present invention, the term “mutation”refers to a deletion, substitution or addition of one or more residues,or a combination of these possibilities.

[0036] According to a first embodiment of the invention, the adenoviralfiber according to the invention derives from a fiber-of a type 5adenovirus (Ad5) comprising all or part of the sequence as shown insequence identifier No. 1 (SEQ ID NO: 1), and is characterized in thatit is modified by mutation of one or more residues of the region betweenresidues 400 and 428 more particularly between residues 404 and 418, andpreferably between residues 404 and 408, of SEQ ID NO: 1. Entirelypreferably, such an adenoviral fiber has the properties (i) and (ii) setout above.

[0037] Preferably, the invention relates to a fiber of a type 5adenovirus, characterized in that the mutated residue is selected fromthe threonine residue at position 404, the alanine residue at position406 and the serine residue at position 408.

[0038] Because of their spatial location in the native fiber, theseresidues are capable of recognizing and/or interacting directly orindirectly with the natural cellular receptor for the adenovirus inquestion.

[0039] According to a particular case of the invention, the mutationproduced is a substitution of at least one amino acid. In this capacity,mention may be made of the following examples of a fiber of a type 5adenovirus, for which:

[0040] the serine residue at position 408 is substituted with a residuehaving at least two carboxyl groups, and in particular with a residueselected from the group consisting of aspartic acid and glutamic acid,and/or the threonine residue at position 404 is substituted with aglycine residue and/or

[0041] the alanine residue at position 406 is substituted with a lysineresidue.

[0042] It is also possible to introduce several substitutions into thetargeted region of the fiber, in particular at the amino acids forming abend, preferable of αα type.

[0043] In accordance with the invention, it is preferable not todrastically modify the three-dimensional structure of the adenoviralfiber; thus, the amino acids forming a bend will be replaced withresidues forming a similar structure, such as those mentioned in Xia etal. (1994).

[0044] The fiber of the present invention can also be modified bydeletion. The region removed can concern all or part of the exposeddomain and, in particular, of loop AB.

[0045] According to an advantageous embodiment, when one at least of themodifications is deletion of at least three consecutive residues of aloop and/or of a pleated sheet, the deleted residues can be replacedwith residues of an equivalent loop and/or pleated sheet derived from afiber of a second adenovirus capable of interacting with a cellularreceptor other than that recognized by the first adenovirus. This makesit possible to maintain the structure of the fiber according to theinvention, while at the same time confering upon it a host specificitycorresponding to that of the second adenovirus. As indicated in Xia etal. (1994), the infection of type 2 and type 5 adenoviruses is differentfrom that of type 3 and type 7 adenoviruses. Thus, the residues deletedfrom an Ad5 or Ad2 fiber deleted of at least three consecutive residuesamong those specified above can be substituted with the residues derivedfrom an equivalent region of the Ad3 or Ad7 fiber, so as to decrease theability of said fiber to bind the receptor for Ad5 and to confer upon ita novel specificity toward the cellular receptor for Ad3 or Ad7.

[0046] The present invention also relates to a fiber of an adenovirushaving a substantially decreased ability to bind to the natural cellularreceptor, as shown above, but nevertheless capable of trimerizing. Sucha property is, in particular, determined using the technique describedin the experimental section of the application.

[0047] According to an equally advantageous embodiment, the fiberaccording to the invention also comprises a ligand. For the purpose ofthe present invention, the term “ligand” defines any entity capable ofrecognizing and binding, preferably with high affinity, a cellularantiligand other than the natural cellular receptor for the nonmutatedadenoviral fiber. This antiligand can be expressed or exposed at thesurface of the cell the targeting of which is desired (cell surfacemarker, receptor, antigenic peptide presented by histocompatibilityantigens, etc.), naturally or subsequent to a modification of saidtarget aimed at making it express or expose such an antiligand at itssurface. In accordance with the aims pursued by the present invention, aligand can be an antibody or an antibody fragment, a lipid, aglycolipid, a hormone, a polypeptide, a polymer (PEG, polylysine, PEI,etc.) or a sugar. The term “antibody” refers, in particular, tomonoclonal antibodies, antibody fragments (such as, for example, Fabfragments) and single chain antibodies (scFv). These names andabbreviations are conventional in the field of immunology.

[0048] In the context of the present invention, it may be advantageousto target more particularly a tumor cell, an infected cell, a specificcell type or a category of cells bearing a specific surface marker. Forexample, if the host cell to be targeted is a cell infected with the HIVvirus (Human Immunodeficiency Virus), the ligand can be a fragment ofantibody against fusin, the CD4 receptor or against an exposed viralprotein (envelope glycoprotein), or the portion of the HIV virus TATprotein stretching from residues 37 to 72 (Fawell et al., 1994, Proc.Natl. Acad. Sci. USA 91, 664-668). If it is a tumor cell, the choicewill relate to a ligand which recognizes a tumor-specific antigen (forexample the MUC-1 protein in the case of breast cancer, or certainepitopes of the HPV papilloma virus E6 or E7 proteins) or which isoverexpressed (IL-2 receptor overexpressed in certain lymphoid tumors).If the intention is to target—T lymphocytes, a T-cell receptor ligandcan be used. Moreover, transferrin is a good candidate for hepatictargeting. In general, the ligands which can be used in the context ofthe invention are widely described in the literature and can be clonedusing standard techniques. It is also possible to synthesize themchemically, and to couple them to the fiber according to the invention.In this respect, the coupling of galactosyl residues should conferhepatic specificity due to the interaction with asialoglycoproteinreceptors. However, the preferred embodiment consists in inserting theligand at the C-terminal end of the fiber according to the invention oras a replacement for the residues deleted when one at least of themodifications is a deletion of at least 3 consecutive residues.

[0049] Another subject of the invention relates to a peptide fragmentcharacterized in that it comprises the region stretching from pleatedsheet A to pleated sheet B, and including loop AB, of a modified fiberas described above. Such a peptide fragment has, in particular, thefollowing properties:

[0050] (i) when this peptide fragment is incorporated in place of aregion stretching from pleated sheet A to pleated sheet B, and includingloop. AB, of a given heterologous adenoviral fiber, the adenoviralparticle comprising said mutated fiber does not substantially attach tothe natural cellular receptors

[0051] (ii) when the adenoviral particle comprising said mutated fiberaccording to (i) also comprises a ligand specific for an antiligand, itis possible to confer upon said modified particle a novel tropism forone or more specific cell types bearing, at their surface, saidantiligand, in comparison with the adenoviral particle which does notcomprise such a mutated fiber.

[0052] The invention relates more specifically to such a peptidefragment characterized in that it is the sequence stretching fromresidue 388 to residue 592 of a fiber of a type 5 adenovirus (Ad5)comprising all or part of the sequence as shown in sequence identifierNo. 1 (SEQ ID NO: 1) and comprising at least one mutation at one or moreresidues of the region between residues 400 and 428.

[0053] The present invention also relates to an adenoviral particlewhich comprises, at its surface, a mutated fiber according to theinvention and, optionally, a ligand as defined above. According to apreferred case, this adenoviral particle lacks a functional nativefiber. The mutated fiber of the invention can be expressed by theadenoviral genome itself, in particular when said adenoviral particlecontains such a genome, or provided in trans by a complementation cellline, such as those defined hereinafter. According to a particularembodiment, the adenoviral particle of the invention is as shown aboveand is characterized in that said ligand is inserted into an adenoviralcapsid protein other than the fiber, in particular the hexon or thepenton.

[0054] According to a particular case of the invention, said adenoviralparticle of the invention is “empty”, i.e. it contains no nucleic acid.The use of such viral particles is in particular illustrated in documentWO 95/21259, mentioned later. When, on the contrary, this adenoviralparticle contains an adenoviral genome, reference will preferably bemade to an adenoviral virus (or adenovirus) and, in the specific case inwhich said genome is also modified, reference will more especially bemade to a recombinant adenoviral virus (or recombinant adenovirus).

[0055] Such cases are described in greater detail hereinafter. Theinvention therefore also relates to such adenoviruses and recombinantadenoviruses.

[0056] According to the invention, said ligand can be chemically coupledto said adenoviral particle. However, preference is given to the variantaccording to which the sequences encoding the ligand are inserted intothe adenoviral genome, and preferably into the sequences encoding themodified fiber according to the invention, and more specifically inframe in order to preserve the reading frame. The insertion can takeplace at any site. However, the preferred insertion site is upstream ofthe stop codon at the C-terminal end, or in place of the deletedresidues. It is also possible to envisage introducing the sequences ofthe ligand into other adenoviral sequences, in particular those encodinganother capsid protein, such as the hexon or the penton.

[0057] Advantageously, the invention relates to a recombinant adenoviruswhich is replication-defective, i.e. incapable of autonomous replicationin a host cell. The deficiency is obtained by a mutation or deletion ofone or more essential viral genes and, in particular, of all or part ofthe E1 region in the adenoviral genome. Deletions in the E3 region canbe envisaged in order to increase cloning capacities. However, it may beadvantageous to conserve the sequences encoding the gp19k protein(Gooding and Wood, 1990, Critical Reviews of Immunology 10, 53-71) inorder to modulate the immune responses of the host. Of course, thegenome of an adenovirus according to the invention can also comprisefurther deletions or mutations affecting other regions, in particularthe E2, E4 and/or L1-L5 regions (see, for example, WO 94/28152 or WO94/12649, or Ensinger et al., 1972, J. Virol. 10, 328-339, describingthe heat-sensitive mutation of the DBP gene of E2).

[0058] According to a preferred embodiment, a recombinant adenovirus ofthe invention comprises one or more gene(s) of interest placed under thecontrol of the elements required for its (their) expression in a hostcell. The gene in question can be of any origin, genomic, cDNA(complementary DNA) or hybrid (minigene lacking one or more introns). Itcan be attained using conventional techniques of molecular biology, orby chemical synthesis. It can encode an antisense RNA, a ribozyme or anmRNA which will then be translated into a polypeptide of interest. Thispolypeptide can be cytoplasmic or membrane-bound, or can be secreted bythe host cell. Moreover, it can be all or part of a polypeptide as foundnaturally, of a chimeric polypeptide originating from the fusion ofsequences of diverse origins, or of a polypeptide which is mutated withrespect to the native sequence and which has improved and/or modifiedbiological properties.

[0059] In the context of the present invention, it may be advantageousto use the genes encoding the following polypeptides:

[0060] cytokines or lymphokines (α-, β- and γ-interferons, interleukins,and in particular IL-2, IL-6, IL-10 or IL-12, tumor necrosis factors(TNFs), colony stimulating factors (GM-CSF, C-CSF, M-CSF, etc.);

[0061] cellular or nuclear receptors, in particular those recognized bypathogenic organisms (viruses, bacteria or parasites), and preferably bythe HIV virus, or their ligands;

[0062] proteins involved in a genetic disease (factor VII, factor VIII,factor IX, dystrophin or minidystrophin, insulin, CFTR (Cystic FibrosisTransmembrane Conductance Regulator) protein, growth hormones (hGH));

[0063] enzymes (urease, renin, thrombin, etc.);

[0064] enzyme inhibitors (α1-antitrypsin, antithrombin III, viralprotease inhibitors, etc.);

[0065] polypeptides with an antitumor effect, capable of inhibiting, atleast partially, the initiation or progression of tumors or cancers(antibodies, inhibitors acting on cell division or on transductionsignals, tumor suppressor gene expression products, for example p53 orRb, proteins which stimulate the immune system, etc.);

[0066] class I or II major histocompatibility complex proteins orregulatory proteins acting on the expression of the corresponding genes;

[0067] polypeptides capable of inhibiting a viral, bacterial orparasitic infection or its development (antigenic polypeptides havingimmunogenic properties, antigenic epitopes, antibodies, transdominantvariants capable of inhibiting the action of a native protein bycompetition, etc.);

[0068] toxins (herpes simplex virus 1 thymidine kinase (TK-HSV-1),ricin, cholera toxin, diptheria toxin, etc.) or immunotoxins; and

[0069] markers (β-galactosidase, luciferase, etc.).

[0070] It should be pointed out that this list is not limiting and thatother genes can also be used.

[0071] Moreover, a recombinant adenovirus according to the invention canalso comprise a selection gene allowing the selection or identificationof the infected cells. Mention may be made of the neo gene (encodingneomycin phosphotransferase) which confers resistance to the G418antibiotic, the dhfr (dihydrofolate reductase) gene, the CAT(chloramphenicol acetyltransferase) gene, the pac(puromycinacetyltransferase) gene or the gpt (xanthine guaninephosphoribosyl transferase) gene. In general, the selection genes areknown to those skilled in the art.

[0072] The expression “elements required for the expression of a gene ofinterest in a host cell” is intended to mean the set of elementsallowing its transcription into RNA and the translation of an mRNA intoprotein. Among these, the promoter is of particular importance. In thecontext of the present invention, it can derive from any gene ofeukaryotic, or even viral, origin and can be constitutive orregulatable. Moreover, it can be modified so as to improve the promoteractivity, suppress a transcription-inhibiting region, make aconstitutive promoter regulatable or vice versa, introduce a restrictionsite, etc. Alternatively, it can be the natural promoter of the gene tobe expressed. Mention may be made, by way of examples, of the CMV(cytomegalovirus), RSV (Rous Sarcoma Virus), HSV-L virus TK gene, SV40virus (Simian Virus 40) early, and MLP adenoviral viral promoters, orthe eukaryotic promoters of the murine or human PGK (phospho glyceratekinase), α1-antitrypsin (liver-specific) and immunoglobulin(lymphocyte-specific) genes.

[0073] Of course, a gene of interest used in the present invention canalso comprise additional elements required for expression (intronicsequence, signal sequence, nuclear localization sequence, transcriptiontermination sequence, translation initiation site of IRES or other type,etc.) or for its persistence in the host cell. Such elements are knownto those skilled in the art.

[0074] The present invention also relates to a DNA fragment encoding afiber or a peptide fragment according to the invention, and also to avector for expressing such a fiber or such a fragment. Any type ofvector can be used for this purpose, whether of plasmid or viral,integrating or nonintegrating, origin. Such vectors are commerciallyavailable or described in the literature. Similarly, those skilled inthe art are capable of adjusting the regulatory elements required forthe expression of the DNA fragment according to the invention. Accordingto one particular case of the invention, a said vector will be anadenoviral vector capable of producing, under suitable culturingconditions, adenoviral particles according to the invention, namelyadenoviruses or recombinant adenoviruses as described above.

[0075] The invention also relates to a process for preparing adenoviralparticles according to the invention, in which:

[0076] the adenoviral genome encoding a modified fiber according to theinvention is transfected into a suitable cell line, for example the 293line;

[0077] said transfected cell line is cultured cultured under suitableconditions so as to allow the production of said adenovirus or of saidrecombinant adenovirus, and

[0078] the empty particles are recovered by purifying the cell lysate ona density gradient, in particular a cesium chloride gradient forexample.

[0079] The empty particles sediment, for example, at 1.3 g/ml of cesiumchloride, while the recombinant adenoviruses (particles containing theAd genome), themselves, sediment at 1.34 g/ml (D'Hallivin, 1995, Cur.Top. Microbiol. Immunol, 199, 47-66).

[0080] According to another process, it is possible to obtain emptyparticles after transfecting an adenoviral genome carrying a modifiedencapsidation sequence, and also containing a DNA fragment encoding amodified fiber according to the invention, into suitable cells. Themodification of the encapsidation region makes it possible to decrease,or even eliminate, the phenomenon of encapsidation of the adenoviralgenome in the particles (Gräble and Hearing, 1992, J. Virol. 66,723-731). The production steps which follow the culturing are identicalto those described above.

[0081] The invention also relates to a process for preparing anadenovirus or a recombinant adenovirus according to the invention,according to which:

[0082] the genome of said adenovirus, which may or may not berecombinant and which may or may not be replication-defective, istransfected into a suitable cell line,

[0083] said transfected cell line is cultured under suitable conditionsso as to allow the production of said adenovirus or of said recombinantadenovirus (it is also possible to refer to adenoviral particles), and

[0084] said adenovirus or said recombinant adenovirus is recovered fromthe culture of said transfected cell line and, optionally, saidadenovirus is purified.

[0085] The choice of cell line depends, where appropriate, on thedeficient functions of the adenovirus according to the invention. Acomplementation line capable of providing the defective function(s), intrans, will in particular be used. The 293 (ATCC CRL 1573) or PERC6(ECACC 96022940) lines are most particularly suitable for complementingthe E1 function (Graham et al., 1977, J. Gen. Virol. 36, 59-72 or WO97/00326, respectively). For an E1, and E2 or E4 double deficiency, acell line among those described in French Patent Application FR 2737222(96 04413) can be used. It is also possible to use an auxiliary virus inorder to complement the defective adenovirus according to the inventionin any host cell, or a mixed system using a complementation cell and anauxiliary virus, in which the elements are dependent upon each other.The means for propagating a defective adenovirus are known to thoseskilled in the art, who can refer, for example, to Graham and Prevec,1991 (Methods in Molecular Biology, vol. 7, p. 190-128; Ed. E. J. Murey,The Human Press Inc.). The adenoviral genome is preferably reconstitutedin vitro in Escherichia coli (E. coli), by ligation or homologousrecombination (see, for example, French Application FR 2727689 (9414470)). The purification processes are described in the state of theart. Mention may be made of the density gradient centrifugationtechnique.

[0086] According to an alternative process, it is also possible toconstruct “empty” adenoviral particles artificially by associatingcarboxy- or amino-terminal ends of adenoviral capsid proteins, peptidesor glycoproteins, with lipids. Such modified lipids, incorporating inparticular the peptide fragments of the invention, can then beincorporated into a liposome. Such a technique has been described byTikchonenko et al., 1988, Gene 63, 321-330 in the case of liposomesbearing, at their surface, influenza virus glycoproteins.

[0087] The present invention also relates to a cell line comprising,either in a form integrated into the genome or in the form of anepisome, a DNA fragment encoding a fiber according to the invention,placed under the control of the elements allowing its expression. Thesaid line can derive from a cell complementing one or more adenoviralfunctions selected from those encoded by the E1, E2, E4 and L1-L5regions. It preferably derives from the 293 line or from the PERC6 line.Such a line can be used for preparing an adenovirus, in particular arecombinant adenovirus, the genome of which lacks all or part of thesequences encoding the fiber (so as to produce a nonfunctional fiber ornot to produce a fiber).

[0088] For this reason, the invention also relates to a process forproducing adenoviral particles containing an adenoviral genome lackingall or part of the sequences encoding a fiber, characterized in that:

[0089] said genome is transfected into a cell line given above,

[0090] said transfected cell line is cultured under suitable conditionsso as to allow the production of said adenoviral particle, and

[0091] said adenoviral particle is recovered from the culture of saidtransfected cell line and, optionally, said adenoviral particle ispurified.

[0092] The present invention also covers a host cell which can beinfected with an adenovirus according to the invention or which can beobtained using a process according to the invention. It isadvantageously a mammalian cell and, in particular, a human cell. It canbe a primary or tumor cell and of any origin, for example hematopoietic(totipotent stem cell, leukocyte, lymphocyte, monocyte or macrophage,etc.), muscle, nasal; pulmonary, tracheal, hepatic, epithelial orfibroblast origin.

[0093] A subject of the invention is also a composition which comprises,as a therapeutic or prophylactic agent, a host cell, an adenoviralparticle or an adenovirus, in particular a recombinant adenovirus,according to the invention, which can be obtained using a processaccording to the invention, in combination with a support which isacceptable from a pharmaceutical point of view. The compositionaccording to the invention is, in particular, intended for thepreventive or curative treatment of diseases such as genetic diseases(hemophilia, cystic fibrosis, diabetes or Duchenne, Becker, etc.myopathy), cancers, such as those induced by oncogenes or viruses, viraldiseases, such as hepatitis B or C and AIDS (acquired immunodeficiencysyndrome resulting from HIV infection), and recurrent viral diseases,such as viral infections caused by the herpesvirus.

[0094] A composition according to the invention can be manufacturedconventionally. In particular, a therapeutically effective amount of thetherapeutic or prophylactic agent is combined with a support which isacceptable from a pharmaceutical point of view. Such a support isnontoxic for the patient. It can be an injectable solution, an isotonicsolution, the pH of which is compatible with use in vivo, a solution ofdextrose, of glycerol, of mannitol, etc. A composition according to theinvention can be administered locally, systemically or by aerosol, inparticular via the intragastric, subcutaneous, intracardiac,intra-muscular, intravenous, intraperitoneal, intratumoral,intrapulmonary, intranasal or intratracheal route. The administrationcan take-place in a single dose or in a dose repeated one or more timesafter a certain period of delay. The suitable route of administrationand dose vary depending on various parameters, for example on theindividual or on the disease to be treated, or on the gene(s) ofinterest to be transferred. In particular, the viral particles accordingto the invention can be formulated in the form of doses of between 10⁴and 10¹⁴ pfu (plaque forming units), advantageously 10⁵ and 10¹³ pfu,and preferably 10⁶ and 10¹² pfu. The formulation can also include anadjuvant or an excipient which is acceptable from a pharmaceutical pointof view.

[0095] The composition according to the invention can also be formulatedin the form of a solid or semi-solid preparation, in particular in theform of a gas, tablet, capsule, powder, gelatin capsule, granule, cream,solution, suppository or aerosol, depending on the route ofadministration selected.

[0096] In the pharmaceutical compositions of the present invention, thecomposition can be formulated with conventional pharmaceutical supports,known to those skilled in the art.

[0097] These supports comprise, in particular, a phrmaceutical vehiclesuch as gelatin, starch, lactose, magnesium stearate, talc, sucrose orgum arabic, or analogues.

[0098] It is also possible to obtain a preparation of gelatin capsulesby mixing the composition with a diluent and pouring the mixtureobtained into soft or hard gelatin capsules.

[0099] A preparation in the form of syrup or of elixir can contain thecomposition together with a sweetener, an antiseptic, and also aflavoring and a suitable colorant.

[0100] The water-dispersible powders or granules can contain thecomposition as a mixture with dispersants or wetting agents, orsuspending agents, as well as with flavor enhancers or sweeteners.

[0101] For rectal administration, use is made of suppositories which areprepared with binders which melt at the rectal temperature, for examplecacao butter or polyethylene glycols.

[0102] The composition can also be formulated in the form ofmicrocapsules, optionally with one or more additive supports.

[0103] A subject of the present invention is also a compositioncharacterized in that it also comprises at least one compound selectedfrom a naked nucleic acid or a nucleic acid combined with at least onecationic compound.

[0104] With a view to their use in vivo, the adenoviral particlesaccording to the invention can also be complexed with synthetic ornatural compounds. Such adenoviral particles, and also their use, are,for example, described in O'Riordan et al., 1999, Human Gene Therapy,10, 1349-1358 or in patent application WO 98/44143. The content of thesedocuments is incorporated into the present application by reference.

[0105] Finally, the present invention relates to the use of a peptidefragment, of an adenoviral particle, of an adenovirus or of a host cellaccording to the invention, or of an adenovirus which can be obtainedusing a process according to the invention, for preparing a medicinalproduct intended for the treatment of the human or animal body.According to a first possibility, the medicinal product can beadministered directly in vivo (for example by intravenous injection,into an accessible tumor, into the lungs by aerosol, etc.). The ex vivoapproach can also be adopted, which consists in removing cells from thepatient (bone marrow stem cells, peripheral blood lymphocytes, musclecells, etc.), transfecting or infecting them in vitro according to thetechniques of the art, and readministering them to the patient.

[0106] The invention also extends to a treatment method according towhich a therapeutically effective amount of an adenovirus or of a hostcell according to the invention is administered to a patient who needssuch a treatment.

EXAMPLES

[0107] The aim of the examples which follow is to illustrate the varioussubjects of the present invention and, consequently, they are in no waylimiting in nature.

[0108] The constructs described below are prepared according to thegeneral techniques of genetic engineering and of molecular cloning,detailed in Maniatis et al., (1989, Laboratory Manual, Cold SpringHarbor, Laboratory Press, Cold Spring Harbor, N.Y.) or according to themanufacturer's recommendations when a commercial kit is used. Thecloning steps using bacterial plasmids are preferably carried out in theE. coli strain 5K (Hubacek and Glover, 1970, J. Mol. Biol., 50, 111-127)or BJ 5183 (Hanahan, 1983, J. Mol. Biol. 166, 557-580). The latterstrain is preferably used for the homologous recombination steps. TheNM522 strain (Strategene) is suitable for propagating the M13 phagevectors. The PCR amplification techniques are known to those skilled inthe art (see, for example, PCR Protocols—A guide to methods andapplications, 1990, edited by Innis, Gelfand, Sninsky and White,Academic Press Inc.). As regards the repair of restriction sites, thetechnique used consists in filling the overhanging 5′ ends using thelarge fragment of E. coli DNA polymerase, I (Klenow). The Ad5 nucleotidesequences are those used in the Genebank databank, under the referenceM73260.

[0109] With regard to the cell biology, the cells are transfectedaccording to standard techniques known to those skilled in the art.Mention may be made of the calcium phosphate technique (Maniatis et al.,above), but any other protocol can also be used, such as the DEAEdextran technique, electroporation, methods based on osmotic shocks,microinjection or methods based on the use of cationic lipids. Withregard to the culturing conditions, they are conventional. In theexamples which follow, use is made of the 293 human line (ATCC CRL 1573)and of the Swiss 3T3 (ATCC CCL92), NR6 (Wells et al., 1990, Science 247,962-964) and NR6-hEGFR (Schneider et al., 1986, Proc. Natl. Acad. Sci.USA 83, 333-336) murine lines. It is understood that other cell linescan also be used.

EXAMPLE 1 Construction of an Adenovirus Having a Host Tropism for CellsExpressing the Receptor for GRP (Gastrin Releasing Peptide)

[0110] A. Insertion of the Sequences Encoding the GRP Ligand (Fiber-GRP)

[0111] The plasmid pTG6593 derives from p poly II (Lathe et al., 1987,Gene 57, 193-201) by the introduction of the complete gene encoding theAd5 fiber, in the form of an EcoRI-SmaI fragment (nucleotides (nt) 30049to 33093). The HindIII-SmaI fragment (nt 31994-33093) is isolated andcloned into M13TG130. (Kieny et al., 1983, Gene 26, 91-99), digestedwith these same enzymes, to give M13TG6526. The latter is subjected tosite-directed mutagenesis using the oligonucleotide oTG7000 (SEQ ID NO:2) (Sculptor in vitro mutagenesis kit, Amersham) in order to introduce alinker encoding a spacer arm of 12 amino acids of sequence PSASASASAPGS.The mutated vector thus obtained, M13TG6527, is subjected to, a secondmutagenesis allowing the introduction of the sequence encoding the 10residues of the GRP peptide (GNHWAVGHLM; Michael et al., 1995, GeneTher. 2, 660-668). The oligonucleotide oTG7001 (SEQ ID NO: 3) is usedfor this purpose. The HindIII-SmaI fragment is isolated from the mutatedphage M13TG6528 and introduced, using the homologous recombinationtechnique (Chartier et al., 1996, J. Virol. 70, 4805-4810), into theplasmid pTG6590 carrying the Ad5 adenoviral gene fragment stretchingfrom nt 27081 to 35935 and linearized with MunI (nt 32825). TheSpeI-ScaI fragment (carrying nt 27082 to 35935 of the Ad5 genome,modified by introducing the spacer arm and the GRP peptide) is isolatedfrom the vector above, referred to as pTG8599, and is then exchangedagainst the equivalent fragment of pTG6591, digested beforehand withthese same enzymes. By way of indication, pTG6591 comprises thewild-type adenoviral sequences from positions 21562 to 35935, pTG4600 isobtained, from which the BstEII fragment is isolated (nt 24843 to35233). After homologous recombination with the plasmid pTG3602 whichcomprises the Ad5 genome (described in greater detail in InternationalApplication WO 96/17070), the vector pTG4601 is generated.

[0112] A cassette allowing the expression of the LacZ gene is introducedin place of the E1 adenoviral region by homologous recombination betweenthe plasmid pTG4061 linearized with ClaI and a BsrGI-PstI fragmentcomprising the LacZ gene encoding β-galactosidase under the control ofthe Ad2 MLP promoter and the SV40 virus polyadenylation signal. Thisfragment is isolated from the vector pTG8526 containing the 5′ end ofthe viral genomic DNA (nt 1 to 6241) in which the E1 region (nt 459 to3328) is replaced with the LacZ expression cassette. Its construction iswithin the scope of those skilled in the art. The final vector isreferred to as pTG4628.

[0113] The corresponding viruses AdTG4601 and AdTG4628 are obtained bytransfecting the adenoviral fragments released from the plasmidsequences by PacI digestion, into the 293 line. By way of indication,AdTG4601 carries the complete Ad5 genome in which the fiber genecomprises, in its 3′ end, a spacer arm followed by the GRP peptide. Therecombinant virus AdTG4628 also carries the cassette for expressing theLacZ reporter gene under the control of the MLP adenoviral promoter.

[0114] B. Study of the Tropism of the Virus Carrying the Fiber-GRP

[0115] The presence of the GRP peptide in the adenoviral fiber makes itpossible to target cells expressing, at their surface, the receptor forGRP. The expression of the messages encoding the latter is studied in293 cells and in Swiss 3T3 murine cells (Zachary et al., 1985, Proc.Natl. Acad. Sci. USA 82, 7616-7620) by Northern blot. A mixture of 2 DNAfragments complementary to the sequence encoding the receptor for GRP,labeled with the ³²P isotope using conventional techniques, is used as aprobe. By way of indication, the fragments are produced by reverse PCRon total cellular RNAs using the oligonucleotides oTG10776 (SEQ ID NO:4) and oTG10781 (SEQ ID NO: 5) (Battey et al., 1991, Proc. Natl. Acad.Sci. USA 88, 395-399; Corjay et al., 1991, J. Biol. Chem. 266,18771-18779). The intensity of the mRNAs detected is much greater in thecase of the Swiss-3T3 cells than in the case of the 293 cells,indicating the overexpression of the GRP receptor by the murine line.

[0116] Competition experiments are carried out on the 2 cell types. Thecompetitor consists of the Ad5 fiber knob produced in E. coli, theadenoviral cellular receptor-binding properties of which have been shown(Henry et al., 1994, J. Virol 68, 5239-5246). The cells in monolayer arepre-incubated for 30 min in the presence of PBS or of increasingconcentrations of recombinant Ad5 knob (0.1 to 100 μg/ml), in DMEMmedium (Gibco BRL) supplemented with 2% fetal calf serum (FCS). Then,the virus AdTG4628, the fiber of which contains the GRP peptide, isadded at a multiplicity of infection of 0.001 infectious units/cell, for24 h at 37° C. By way of control, and according to the same experimentalconditions, the recombinant virus AdLacZ (Stratford-Perricaudet et al.,1992, J. Clin. Invest. 90, 626-630), which carries a native fiber gene,is used. The cells are then fixed and the expression of the LacZ gene isevaluated (Sanes et al., 1986, EMBO J. 5, 3133-3142). The number of bluecells is representative of the efficiency of the viral infection.Competition inhibition causes a decrease in the number of colored cellswith respect to a noninfected control (PBS).

[0117] The addition of recombinant Ad5 knob at a concentration of 100μg/ml strongly inhibits the infection of the 293 cells with the virusesAdLacZ and AdTG4628 (degree of inhibition of 95 and 98%). This suggeststhat the presence of the competitor prevents the interaction of theadenoviral fiber with its natural cellular receptor. On the other hand,the two viruses behave differently on the Swiss-3T3 cells. The infectionof the virus AdTG4628 in the presence of 100 μg/ml of competitor is onlypartially inhibited, whereas, under the same experimental conditions,that of the virus AdLacZ having the native fiber is totally inhibited.These results suggest that the infection of the Swiss-3T3 cells withAdTG4628 is, in part, mediated by an independent receptor, probably theGRP receptor which these cells overexpress. In conclusion, the additionof the GRP ligand to the C-terminal end of the fiber promotes theinfection of the cells expressing the GRP receptor, independently of thefiber-natural cellular receptor interaction.

EXAMPLE 2 Construction of an Adenovirus Having a Tropism for Tumor CellsExpressing Mucins

[0118] Construction insertion of the EPPT peptide, as described in U.S.Pat. No. 5,591,593, into the C-term of the fiber. This modificationconfers binding to mucins overexpressed on tumor cells.

[0119] OTG11992: SEQ ID NO 12

[0120] mutagenesis with m13TG6527 to give m13TG6572. Homologousrecombination with pTG4213 to give pTG4278.

EXAMPLE 3 Construction of an adenovirus having a tropism for tumor cellsexpressing α₄β₁ Integrins

[0121] Construction: insertion of the LDV peptide, as described in U.S.Pat. No. 5,628,979, in the C-term of the fiber. This modificationconfers binding to α₄β₁ integrins overexpressed on tumor cells.

[0122] OTG 1191: SEQ ID NO 13

[0123] mutagenesis with m3TG6527 to give M13TG13265.

EXAMPLE 4 Construction of an Adenovirus Having a Host Tropism for CellsExpressing the EGF (Epidermal Growth Factor) Receptor

[0124] This example describes a fiber carrying the EFG sequences at itsC-terminal end. For this, the oligonucleotides oTG11065 (SEQ ID NO: 6),and oTG11066 (SEQ ID NO: 7) are used to amplify a HindIII-XbaI fragmentfrom the plasmid M13TG6527. The oligonucleotides oTG11067 (SEQ. ID NO:8), and oTG11068 (SEQ ID NO: 9) make it possible to generate anXhoI-SmaI fragment (ranging from the stop codon up to nt 33093) fromM13TG6527. The complementary DNA of EGF, obtained from the ATCC(#59957), is amplified in the form of an XhoI-XbaI fragment using theoligonucleotides oTG 11069 (SEQ ID NO: 10) and oTG11070(SEQ ID NO: 11).The 3 fragments digested with the appropriate enzymes are then religatedto give a HindIII-SmaI fragment containing EGF fused to the C-terminalend of the fiber. The same procedure of homologous recombination as thatdescribed in example 1 is used to reposition this fragment in itsgenomic context.

[0125] However, the cloning steps can be simplified by introducing aunique BstBI site into the targeted region using conventionalmutagenesis techniques. pTG4609 is obtained. The homologousrecombination between pTG4609 linearized with BstBI and the HindIII-SmaIfragment above generates the plasmid pTG4225 carrying the wild-type Elregion. Its equivalent carrying the LacZ expression cassette, pTG4226 isobtained by homologous recombination with the pTG4213 digested withBstBI. The viruses AdTG4225 and AdTG4226 can be produced conventionallyby transfecting a suitable cell line, for example, overexpressing thereceptor for EGF.

[0126] In order to test the specificity of infection of these viruses,NR6 murine fibroblastic cells and Nr6-hEGFR cells expressing thereceptor for human EGF can be used. Competition with the recombinant Ad5knob or with EGF makes it possible to evaluate the involvement of theEGF or natural cellular receptors in mediating the infection of theviruses.

EXAMPLE 5 Modifications of the Fiber Knob so as to Eliminate the Bindingto the Natural Cellular Receptor

[0127] A. Modifications of the Fiber Sequences

[0128] The mutation of region AB (amino acid 404-418), of the adenoviralfiber was undertaken in order to eliminate the ability of the fiber tobind its natural receptor, and the addition of a ligand will make itpossible to modify the tropism of the corresponding adenoviruses.

[0129] replacement, in loop AB, of the serine at position 408 with theglutamic acid residue of serotype 3 using the oligo oTG12499 (SEQ ID NO:14);

[0130] replacement, in loop AB, of the alanine at position 406 with thelysine residue of serotype 3 using the oligo oTG12498 (SEQ ID NO: 15);

[0131] replacement, in loop AB, of the threonine at position 404 withthe glycine residue of serotype 3 using the oligo oTG12740 (SEQ ID NO:16).

[0132] The mutageneses can be carried out on the vector M13TG6526 orM13TG6528. The first carries the wild-type HindIII-SmaI fragment and thesecond carries the same fragment modified by inserting the GRPsequences. The plasmids carrying the adenoviral genome can bereconstituted as described previously for the plasmids pTG4225(wild-type E1) and pTG4226 (LacZ in place of the E1 region) (byhomologous recombination with the plasmid pTG4609 or pTG4213). Theviruses are generated by transfecting 293 cells, 293 cells expressingthe wild-type fiber (Legrand et al., 1999; J. Virol., 73, 907-919) orcells overexpressing the receptor which binds the ligand in question.Such cells can be generated by transfection of the correspondingcomplementary DNA. Cells which do not naturally express the naturalcellular receptor for adenoviruses, for example the Daudi line (ATCCCCL213) are preferably used. Oligo M13 Plasmid Mutation oTG- M13TG pTG-ABloop (404-418): 404TPAPS408 404GPAPS408 12740 14017 14283 404TPKPS40812498 6587 4289 404TPAPE408 12499 6588 4291

[0133] B. Study of the Incorporation of the Modified Fiber into theViral Particle and of its Use in the Entry of the CorrespondingAdenovirus

[0134] In order to be sure that the mutated viruses indeed carry themodified fiber proteins in their capsid, the viruses purified afteramplification in the 293 cells are loaded onto 10% acrylamide gel underdenaturing conditions (SDS-PAGE). The various proteins are detected bysilver nitrate staining. Alternatively, the fiber is revealedspecifically by carrying out a western blot using a serum directedagainst the Ad5 fiber knob (Henry et al., 1994, above). A strong signalwith the expected size indicates that the viruses incorporatestoichiometric amounts of the protein of interest. Given that only thetrimeric fiber is capable of binding the penton base (Novelli andBoulanger, 1991, above) and of being incorporated into the particle, thedetection of the protein in the experiment above indicates that themodified fiber is still capable of forming trimers.

[0135] Use of the modified fiber to allow the entry of the correspondingmutated virus can be studied by carrying out the competition experimentsusing recombinant knob as described above in Example 1B. An efficientinfection in the presence of saturating concentrations of the wild-typepeptide indicates an infection independent of the binding to the naturalprimary receptors. This suggests a greatly decreased affinity of themodified fiber for its receptors.

EXAMPLE 6 Insertion of the Ligand Into a Capsid Protein Other than theFiber, in Combination with One of the Abovementioned Modifications ofthe Fiber

[0136] This example describes the insertion of the EGF ligand into thehexon capsid protein. Of course, it is preferable for the correspondingadenovirus to have lost its ability to attach to the natural cellularreceptor. Its genome can, for example, include a modified fiber gene, orlack a portion, at least, of the fiber sequences.

[0137] A transfer plasmid for the homologous recombination covering theregion of the Ad5 genome encoding the hexon (nt 18842-21700) isconstructed. The HindIII-XhoI fragment of Ad5 (nt 18836-24816) is clonedinto pBSK+ (Stratagene) digested with these same enzymes, to give theplasmid pTG4224. The sequences encoding the EGF peptide are introducedinto the L1 hypervariable loop of the hexon by creating chimericfragments using PCR: hexon (nt19043-19647)-XbaI-EGF-BsrGI-hexon(nt19699-20312). The nt19043 to 19647 fragment is obtained by PCRamplification using the plasmid pTG3602 with the oligonucleotidesoTG11102 (SEQ ID NO: 17) and oTG11103 (SEQ ID NO: 18). The nt19699 to20312 fragment is amplified from the same DNA with the oligonucleotidesoTG11104 (SEQ ID NO: 19) and oTG11105 (SEQ ID NO: 20). The EGF is clonedusing the cDNA with the aid of the oligonucleotides oTG11106 (SEQ ID NO:21) and oTG11107 (SEQ ID NO: 22) allowing the EGF coding sequence to beplaced in frame with the hexon. The PCR products are digested with theappropriate enzymes and then religated. The chimeric fragment can thenbe inserted by homologous recombination into the plasmid pTG4224linearized with NdeI (nt 19549), to give pTG4229. The sequences encodingthe modified hexon can be obtained by HindIII-XhoI digestion andrepositioned in their genomic context by homologous recombination. Usemay be made of the vector pTG3602, pTG4607 or pTG4629 linearized withSgfI, or a vector carrying the adenoviral genome deleted of the fibersequences (such as pTG4607 described above) or expressing a modifiedfiber.

[0138] The adenoviral genome incapable of producing a functional nativefiber is obtained through a deletion which affects the initiator codon,but which does not extend to the other adenoviral ORFs. The following iscarried out: the adenoviral fragment 5′ of the deletion (nt 30564 to31041) is amplified by PCR using the primers oTG7171 and oTG7275 (SEQ IDNO: 23 and 24). The amplification of the fragment positioned 3′ (nt31129 to 33099) uses the primers oTG7276 and oTG7049 (SEQ ID NO: 25 and26). The PCR fragments are digested with XhoI and ligated before beingintroduced by homologous recombination into the vector pTG6591linearized with NdeI, to give pTG4602. Then, the BstEII fragmentisolated from the latter is subjected to homologous recombination withthe vector pTG3602 digested with SpeI. pTG4607 is obtained. The vectorpTG4629 is equivalent to pTG4607, but also carries the LacZ expressioncassette in place of E1.

[0139] The corresponding viruses can be obtained after transfecting 293cells or 293 cells expressing the wild-type fiber (Legrand et al., 1999,above), or cells overexpressing the receptor for EGF. The study of thespecificity of infection may be carried out as described previously,using EGF as a competitor.

1 28 1 581 PRT Mastadenovirus 5 Ad5 fiber Position on the map 31063 to33120 of the Ad5 genome. 1 Met Lys Arg Ala Arg Pro Ser Glu Asp Thr PheAsn Pro Val Tyr Pro 1 5 10 15 Tyr Asp Thr Glu Thr Gly Pro Pro Thr ValPro Phe Leu Thr Pro Pro 20 25 30 Phe Val Ser Pro Asn Gly Phe Gln Glu SerPro Pro Gly Val Leu Ser 35 40 45 Leu Arg Leu Ser Glu Pro Leu Val Thr SerAsn Gly Met Leu Ala Leu 50 55 60 Lys Met Gly Asn Gly Leu Ser Leu Asp GluAla Gly Asn Leu Thr Ser 65 70 75 80 Gln Asn Val Thr Thr Val Ser Pro ProLeu Lys Lys Thr Lys Ser Asn 85 90 95 Ile Asn Leu Glu Ile Ser Ala Pro LeuThr Val Thr Ser Glu Ala Leu 100 105 110 Thr Val Ala Ala Ala Ala Pro LeuMet Val Ala Gly Asn Thr Leu Thr 115 120 125 Met Gln Ser Gln Ala Pro LeuThr Val His Asp Ser Lys Leu Ser Ile 130 135 140 Ala Thr Gln Gly Pro LeuThr Val Ser Glu Gly Lys Leu Ala Leu Gln 145 150 155 160 Thr Ser Gly ProLeu Thr Thr Thr Asp Ser Ser Thr Leu Thr Ile Thr 165 170 175 Ala Ser ProPro Leu Thr Thr Ala Thr Gly Ser Leu Gly Ile Asp Leu 180 185 190 Lys GluPro Ile Tyr Thr Gln Asn Gly Lys Leu Gly Leu Lys Tyr Gly 195 200 205 AlaPro Leu His Val Thr Asp Asp Leu Asn Thr Leu Thr Val Ala Thr 210 215 220Gly Pro Gly Val Thr Ile Asn Asn Thr Ser Leu Gln Thr Lys Val Thr 225 230235 240 Gly Ala Leu Gly Phe Asp Ser Gln Gly Asn Met Gln Leu Asn Val Ala245 250 255 Gly Gly Leu Arg Ile Asp Ser Gln Asn Arg Arg Leu Ile Leu AspVal 260 265 270 Ser Tyr Pro Phe Asn Ala Gln Asn Gln Leu Asn Leu Arg LeuGly Gln 275 280 285 Gly Pro Leu Phe Ile Asn Ser Ala His Asn Leu Asp IleAsn Tyr Asn 290 295 300 Lys Gly Leu Tyr Leu Phe Thr Ala Ser Asn Asn SerLys Lys Leu Glu 305 310 315 320 Val Asn Leu Ser Thr Ala Lys Gly Leu MetPhe Asn Ala Thr Ala Ile 325 330 335 Ala Ile Asn Ala Gly Asp Gly Leu GluPhe Gly Ser Pro Asn Ala Pro 340 345 350 Asn Thr Asn Pro Leu Lys Thr LysIle Gly His Gly Leu Glu Phe Asp 355 360 365 Ser Asn Lys Ala Met Val ProLys Leu Gly Thr Gly Leu Ser Phe Asp 370 375 380 Ser Thr Gly Ala Ile ThrVal Gly Asn Lys Asn Asn Asp Lys Leu Thr 385 390 395 400 Leu Trp Thr ThrPro Ala Pro Ser Pro Asn Cys Arg Leu Asn Ala Glu 405 410 415 Lys Asp AlaLys Leu Thr Leu Val Leu Thr Lys Cys Gly Ser Gln Ile 420 425 430 Leu AlaThr Val Ser Val Leu Ala Val Lys Gly Ser Leu Ala Pro Ile 435 440 445 SerGly Thr Val Gln Ser Ala His Leu Ile Ile Arg Phe Asp Glu Asn 450 455 460Gly Val Leu Leu Asn Asn Ser Phe Leu Asp Pro Glu Tyr Trp Asn Phe 465 470475 480 Arg Asn Gly Asp Leu Thr Glu Gly Thr Ala Tyr Thr Asn Ala Val Gly485 490 495 Phe Met Pro Asn Leu Ser Ala Tyr Pro Lys Ser His Gly Lys ThrAla 500 505 510 Lys Ser Asn Ile Val Ser Gln Val Tyr Leu Asn Gly Asp LysThr Lys 515 520 525 Pro Val Thr Leu Thr Ile Thr Leu Asn Gly Thr Gln GluThr Gly Asp 530 535 540 Thr Thr Pro Ser Ala Tyr Ser Met Ser Phe Ser TrpAsp Trp Ser Gly 545 550 555 560 His Asn Tyr Ile Asn Glu Ile Phe Ala ThrSer Ser Tyr Thr Phe Ser 565 570 575 Tyr Ile Ala Gln Glu 580 2 60 DNAArtificial Sequence Primer. Synthetic oligonucleotide oTG7000 (codes forPSASASASAPGS) 2 aacgattctt tagctgccgg gagcagaggc ggaggcggag gcgctgggttcttgggcaat 60 3 57 DNA Artificial Sequence Primer. Syntheticoligonucleotide oTG7001 (codes for GRP). 3 aacgattctt tacatcaggtggcccacagc ccagtggttt ccgctgccgg gagcaga 57 4 20 DNA Artificial SequencePrimer. Synthetic oligonucleotide oTG10776. 4 ccttccacgg gaagattgta 20 520 DNA Artificial Sequence Primer. Synthetic oligonucleotide oTG10781. 5ggggtgtctg tcttcacact 20 6 26 DNA Artificial Sequence Primer. Syntheticoligonucleotide oTG11065. 6 gggaagcttg aggttaacct aagcac 26 7 28 DNAArtificial Sequence Primer. Synthetic oligonculeotide oTG11066. 7gggtctagag ctgccgggag cagaggcg 28 8 29 DNA Artificial Sequence Primer.Synthetic oligonucleotide oTG11067. 8 gggctcgagt tatgtttcaa cgtgtttat 299 24 DNA Artificial Sequence Primer. Synthetic oligonucleotide oTG11068.9 gtgcccgggg agtttattaa tatc 24 10 31 DNA Artificial Sequence Primer.Synthetic oligonucleotide oTG11069 (EGF cloning) derived from Homosapiens. 10 gcgtctagaa atagtgactc tgaatgtccc c 31 11 46 DNA ArtificialSequence Primer. Synthetic oligonucleotide oTG11070 (EGF cloning)derived from Homo sapiens. 11 gcgctcgagc acaaacgatt ctttagcgcagttcccacca cttcag 46 12 72 DNA Artificial Sequence Primer. Syntheticoligunucleotide oTG11992. 12 cataacacaa acgattcttt atgttcgtgt tggtggttctcgagcgcaat agctgccggg 60 agcagaggcg ga 72 13 57 DNA Artificial SequencePrimer. Synthetic oligonucleotide oTG11991. 13 cataacacaa acgattctttaatatacgtc tagatagctg ccgggagcag aggcgga 57 14 42 DNA ArtificialSequence Primer. Synthetic oligonucleotide oTG12499 derived fromMastadenovirus. 14 gcatttagtc tacagttagg ctctggagct ggtgtggtcc ac 42 1539 DNA Artificial Sequence Primer. Synthetic oligonucleotide oTG12498derived from Mastadenovirus. 15 gtctacagtt aggagatggc tttggtgtggtccacaaag 39 16 47 DNA Artificial Sequence Primer. Syntheticoligonucleotide oTG12740 derived from Mastadenovirus. 16 ctacagttaggagatggagc gggcccggtc cacaaagtta gcttatc 47 17 23 DNA ArtificialSequence Primer. Synthetic oligonucleotide oTG 11102 (hexon cloning)derived from Mastadenovirus. 17 cggttcatcc ctgtggaccg tga 23 18 38 DNAArtificial Sequence Primer. Synthetic oligonucleotide oTG11103 (hexoncloning) derived from Mastadenovirus. 18 ggcctctaga gttgagaaaaattgcatttc cacttgac 38 19 23 DNA Artificial Sequence Primer. Syntheticoligonucleotide oTG11104 (hexon cloning) derived from Mastadenovirus. 19ggtattgtac agtgaagatg tag 23 20 23 DNA Artificial Sequence Primer.Synthetic oligonucleotide oTG11105 derived from Mastadenovirus. 20cgttggaagg actgtacttt agc 23 21 38 DNA Artificial Sequence Primer.Synthetic oligonucleotide oTG11106 (cDNA EGF cloning) derived from Homosapiens. 21 cgcgtctaga ggcgaatagt gactctgaat gtcccctg 38 22 45 DNAArtificial Sequence Primer. Synthetic oligonucleotide oTG11107 (cDNA EGFcloning) derived from Homo sapiens. 22 ccactgtaca ataccacttt agggcgcagttcccaccact tcagg 45 23 21 DNA Artificial Sequence Primer. Syntheticoligonucleotide oTG7171 (deletion of the fiber) derived fromMastadenovirus. 23 atggttaact tgcaccagtg c 21 24 27 DNA ArtificialSequence Primer. Synthetic oligonucleotide oTG7275 (deletion of thefiber) derived from Mastadenovirus. 24 gggctcgagc tgcaacaaca tgaagat 2725 27 DNA Artificial Sequence Primer. Synthetic oligonucleotide oTG7276(deletion of the fiber) derived from Mastadenovirus. 25 ccgctcgagactcctccctt tgtatcc 27 26 20 DNA Artificial Sequence Primer. Syntheticoligonucleotide oTG7049 (deletion of the fiber) derived fromMastadenovirus. 26 ctgcccggga gtttattaat 20 27 42 DNA ArtificialSequence Primer. Synthetic oligonucleotide oTG7416 (deletion of pleatedsheet H) derived from Mastadenovirus. 27 tgtttcctgt gtaccgttggatcctttagt tttgtctccg tt 42 28 64 DNA Artificial Sequence Primer.Synthetic oligonucleotide oTG10352 (pleated sheet H5 to H3) derived fromMastadenovirus. 28 tgtttcctgt gtaccgttta gcatcacggt cacctcgagaggtttagttt tgtctccgtt 60 taag 64

1. Modified fiber of an adenovirus, comprising at least one mutation atone or more residues within the region of said fiber stretching frompleated sheet A to pleated sheet B, and including loop AB.
 2. Fiber ofan adenovirus according to claim 1, characterized in that it comprisesat least one mutation at one or more residues within loop AB.
 3. Fiberof an adenovirus according to claim 1 or 2, characterized in that itallows, when it is included in a viral particle, the production of asaid viral particle having the following properties: (i) said adenoviralparticle does not substantially attach to the natural cellularreceptors; (ii) when said adenoviral particle also comprises a ligandspecific for an antiligand, said modified particle has a novel tropismfor one or more specific cell types carrying, at their surface, saidantiligand.
 4. Fiber of an adenovirus according to one of claims 1 to 3,characterized in that it derives from a fiber of a type 5 adenovirus(Ad5) comprising all or part of the sequence as shown in sequenceidentifier No. 1 (SEQ ID NO: 1), and in that it comprises at least onemutation at one or more residues of the region between residues 400 and428.
 5. Fiber of a type 5 adenovirus according to claim 4, characterizedin that it comprises at least one mutation at one or more residues ofthe region between residues 404 and 418 of SEQ ID NO:
 1. 6. Fiber of atype 5 adenovirus according to claim 5, characterized in that itcomprises at least one mutation at one or more residues of the regionbetween residues 404 and 408 of SEQ ID NO:
 1. 7. Fiber of a type 5adenovirus according to claim 6, characterized in that said residue isselected from the threonine residue at position 404, the alanine residueat position 406 and the serine residue at position
 408. 8. Fiber of atype 5 adenovirus according to claim 7, characterized in that itcomprises substitution of the serine residue at position 408 with anamino acid residue having at least two carboxyl groups.
 9. Fiber of atype 5 adenovirus according to claim 8, characterized in that saidresidue is selected from the group consisting of aspartic acid andglutamic acid.
 10. Fiber of a type 5 adenovirus according to claim 7,characterized in that it comprises substitution of the threonine residueat position 404 with a glycine residue and/or substitution of thealanine residue at position 406 with a lysine residue.
 11. Fiber of anadenovirus according to one of claims 1 to 10, characterized in that oneat least of the mutations is deletion of at least 3 consecutive residuesof a loop and/or of a pleated sheet of said region.
 12. Fiber of anadenovirus according to claim 11, characterized in that said deletedresidues are replaced with residues of an equivalent loop and/or pleatedsheet derived from a fiber of a second adenovirus of heterologous type,capable of interacting with a cellular receptor other than thatrecognized by said first adenovirus.
 13. Fiber of an adenovirusaccording to one of claims 1 to 12, characterized in that it alsocomprises one or more mutations in: (i) loops CD, DG, GH, HI and/or IJand/or (ii) pleated sheets C, D, G, H, I and/or J.
 14. Fiber of anadenovirus according to one of claims 1 to 13, characterized in that italso comprises a ligand capable of recognizing a cellular antiligandother than the natural cellular receptor of the nonmutated fiber. 15.Fiber of an adenovirus according to claim 14, characterized in that theligand is selected from the group consisting of an antibody or anantibody fragment, a peptide, a lipid, a glycolipid, a hormone, apolymer or a sugar.
 16. Fiber of an adenovirus according to claim 14 or15, characterized in that the ligand is inserted at the C-terminal endof the fiber.
 17. Fiber of an adenovirus according to claim 14 or 15,characterized in that the ligand is inserted as a replacement fordeleted residues.
 18. Peptide fragment characterized in that itcomprises the region stretching from pleated sheet A to pleated sheet B,and including loop AB, of a fiber according to any one of claims 1 to17.
 19. Peptide fragment according to claim 18, characterized in that itis the sequence stretching from residue 388 to residue 592 of a fiberaccording to any one of claims 4 to
 17. 20. DNA fragment or expressionvector encoding a fiber of an adenovirus according to one of claims 1 to17, or a peptide fragment according to either of claims 18 and
 19. 21.Cell line characterized in that it comprises, either in a formintegrated into the genome or in episome form, a DNA fragment accordingto claim 20, placed under the control of the elements allowing itsexpression in said cell line.
 22. Cell line according to claim 21,characterized in that it is also capable of complementing an adenovirusdeficient for one or more functions selected from the functions encodedby the E1, E2, E4 and L1-L5 regions.
 23. Cell line according to claim 21or 22, characterized in that it is produced using the 293 line.
 24. Cellline according to claim 21 or 22, characterized in that it is producedusing the PERC6 line.
 25. Adenoviral particle characterized in that itlacks a functional native fiber, and in that it comprises a fiberaccording to one of claims 1 to
 17. 26. Adenoviral particlecharacterized in that it lacks a functional native fiber, and in that itcomprises a fiber according to one of claims 1 to 17 and a ligandcapable of recognizing a cellular antiligand other than the naturalcellular receptor for said particle.
 27. Adenoviral particle accordingto claim 26, characterized in that said ligand is inserted into anadenoviral capsid protein other than the fiber, in particular the hexonor the penton.
 28. Adenoviral particle according to one of claims 25 to27, characterized in that it is empty.
 29. Adenoviral particle accordingto one of claims 25 to 27, characterized in that it contains anadenoviral genome.
 30. Adenoviral particle according to claim 29,characterized in that said adenoviral genome is a replication-defectiverecombinant adenoviral genome.
 31. Process for producing an adenoviralparticle according to claim 29, characterized in that: (i) a saidreplication-defective recombinant adenoviral genome is transfected intoa suitable cell line, (ii) said transfected cell line is cultured undersuitable conditions so as to allow the production of said adenoviralparticle, and (iii) said adenovirus is recovered from the culture ofsaid transfected cell line and, optionally, said adenoviral particle ispurified.
 32. Process for producing an adenoviral particle containing anadenoviral genome lacking all or part of the sequences encoding a fiber,characterized in that: said genome is transfected into a cell lineaccording to one of claims 21 to 24, said transfected cell line iscultured under suitable conditions so as to allow the production of saidadenoviral particle, and said adenoviral particle is recovered from theculture of said transfected cell line and, optionally, said adenoviralparticle is purified.
 33. Composition which comprises an adenoviralparticle according to one of claims 25 to 30, or which can be obtainedusing a process according to claim 31 or 32, in combination with asupport which is acceptable from a pharmaceutical point of view. 34.Composition according to claim 33, characterized in that it alsocomprises at least one compound selected from a naked nucleic acid or anucleic acid combined with at least one cationic compound.
 35. Use of anadenoviral particle according to one of claims 25 to 30, or which can beobtained using a process according to claim 31 or 32, for preparing amedicinal product intended for the treatment of the human or animalbody.